Synthesis, Two-photon Fluorescence Imaging and Photodynamic Therapy of Near Infrared Thienyl-BODIPY Photosensitizer

doi:10.6023/A22040141

Abstract

In this paper, two novel thienyl boron dipyrromethene (thienyl-BODIPY) near infrared (NIR) photosensitive dyes, ITBDP-1 and ITBDP-2 were designed and synthesized. The absorption and emission wavelengths of two photosensitizers reach near infrared region. The absorption and emission peaks of ITBDP-1 are 617 and 650 nm while the absorption and emission peaks of ITBDP-2 are 687 and 731 nm respectively. The singlet oxygen yields (Φ_Δ) of photosensitizers were determined using 1,3-diphenyliso- benzofuran (DPBF) as singlet oxygen scavenger. The results turned out that two photosensitizers both could generate singlet oxygen under 660 nm light irradiation, and the absorption of DPBF decreased over 50% within 1 min. Φ_Δof ITBDP-1 and ITBDP-2 are 51% and 24% respectively. Excited state energy level of photosensitizers were studied by density functional theory (DFT) calculation. Theoretical calculation indicates that ITBDP-1 and ITBDP-2 can reach triplet state through intersystem crossing (ISC) after being excited to singlet state, thus improving singlet oxygen yield. ITBDP-1 and ITBDP-2 showed satisfying imaging effect in A549 cells, and ITBDP-1 could show distinct two-photon fluorescence in zebrafish under 900 nm excitation. Apart from good cell imaging effect, these two photosensitizers showed abilities to generate reactive oxygen species (ROS) in tumor cells as well. It can be proved that photosensitizers could produce singlet oxygen in tumor cells and zebrafish under light excitation with dichlorodihydrofluorescein diacetate (DCFH-DA) as ROS indicator. Besides, ITBDP-1 and ITBDP-2 also presented high cytotoxicity under 660 nm light irradiation. In the tetrazolium (MTT) experiment, the maximum half inhibitory concentration (IC₅₀) of ITBDP-1 and TBDP-2 were 2.22 and 2.86 μmol•L^-1 respectively, and the cell viability was over 80% without irradiation, which proved that both photosensitizers possess high phototoxicity and good biocompatibility. ITBDP-1 and ITBDP-2 could realize imaging-guided photodynamic therapy under excitation of NIR light in tumor cells, and displayed two-photon fluorescence imaging in vivo. Photosensitizers ITBDP-1 and ITBDP-2 are excepted to lay good foundation for the application of thienyl-BODIPY photosensitizers in two-photon photodynamic therapy under NIR excitation.

Key words: near infrared photosensitizer, thienyl-BODIPY, photodynamic therapy, two-photon imaging

Cite this article

Badi Liu, Chengjun Wang, Ying Qian. Synthesis, Two-photon Fluorescence Imaging and Photodynamic Therapy of Near Infrared Thienyl-BODIPY Photosensitizer[J]. Acta Chimica Sinica, 2022, 80(8): 1071-1083.

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Fig. & Tab. 16

Scheme 1. (a) Different types of reported photosensitizers; (b) NIR photosensitizer ITBDP-1, ITBDP-2 and their imaging-guided photodynamic therapy

Figure 1. The absorption and emission spectra of thienyl-BODIPY compounds in dichloromethane (all concentrations are 10-5 mol•L-1). (a) UV-Vis and fluorescence emission spectra of ITBDP-1 (10-5 mol•L-1); (b) UV-Vis and fluorescence emission spectra of ITBDP-2; (c) UV-Vis and fluorescence emission spectra of TBDP; (d) UV-Vis and fluorescence emission spectra of ITBDP

Scheme 2. Synthesis routine of NIR thienyl-BODIPY photosensitizers ITBDP-1 and ITBDP-2

Figure 2. (a) Time-dependent plot of UV-Vis intensity of DPBF in ITBDP-1 solution; time-dependent plot of (A/A0) value of DPBF at 414 nm in ITBDP-1 solution and methylene blue solution; (b) time-dependent plot of UV-Vis intensity of DPBF in ITBDP-2 solution; time-dependent plot of (A/A0) value of DPBF at 414 nm in ITBDP-2 solution and methylene blue solution

Compound	$\lambda _{\max }^{\text{abs}}$/ nm	$\varepsilon _{\max }^{\text{abs}}$/(10⁴L•mol^-1•cm^-1)	$\lambda _{\max }^{em}$/nm	Φ_Δ	IC₅₀/ (μmol•L^-1)
ITBDP-1	617	0.62	650	0.51	2.22
ITBDP-2	687	7.60	731	0.24	2.86
TBDP	513	3.50	523	—	—
ITBDP	546	3.90	562	0.63	—

Table 1. Photophysical properties of thienyl-BODIPY derivatives

Figure 3. Ground state structure optimization based on DFT//B3LYP/6-31G(d) and DFT//B3LYP/6-31G(d)/LANL2DZ. (a) calculated excited state energy and frontier orbital diagram of ITBDP-1; (b) calculated excited state energy and frontier orbital diagram of ITBDP-2 based on TD-DFT//cam-B3LYP/6-31G(d) and TD-DFT// cam-B3LYP/6-31G(d)/LANL2DZ

Figure 4. Two-photon fluorescence imaging of ITBDP-1 (2 μmol•L-1, λex=900 nm) incubated with zebrafish for 2 h: (a) head imaging of zebrafish; (b) body imaging of zebrafish; (c) tail imaging of zebrafish

Figure 5. (a1~a3) Fluorescence imaging of ITBDP-1 (2 μmol•L-1) in A549 cells; (b1~b3) fluorescence imaging of ITBDP-2 (2 μmol•L-1) in A549 cells (a1~a3) Fluorescence imaging of ITBDP-1 (2 μmol•L-1) in A549 cells; (b1~b3) fluorescence imaging of ITBDP-2 (2 μmol•L-1) in A549 cells

Figure 6. (a) Cell viability of A549 cells treated with ITBDP-1 under light irradiation for 15 min (660 nm, 4 mW•cm-2) and dark conditions; (b) cell viability of A549 cells treated with ITBDP-2 under light irradiation for 15 min (660 nm, 4 mW•cm-2) and dark conditions

Figure 7. (a) Cell growth curve of A549 cells treated with ITBDP-1 under light irradiation for 15 min (660 nm, 4 mW•cm-2); (b) cell growth curve of A549 cells treated with ITBDP-2 under light irradiation for 15 min (660 nm, 4 mW•cm-2)

Figure 8. ROS imaging of ITBDP-1 in A549 cells. (a1~a4) A549 cells incubated in dark for 3 h in the presence of DCFH-DA and ITBDP-1; (b1~b4) A549 cells incubated in dark for 3 h in the presence of DCFH-DA and ITBDP-1, then irradiated under light (660 nm, 4 mW•cm-2) for 10 min

Figure 9. ROS imaging of ITBDP-2 in A549 cells. (a1~a4) A549 cells incubated in dark for 3 h in the presence of DCFH-DA and ITBDP-2; (b1~b4) A549 cells incubated in dark for 3 h in the presence of DCFH-DA and ITBDP-2, then irradiated under light (660 nm, 4 mW•cm-2) for 10 min; (c1~c4) A549 cells incubated in dark for 3 h in the presence of DCFH-DA and ITBDP-2, then irradiated under light (660 nm, 4 mW•cm-2) for 30 min

Figure 10. ROS imaging of ITBDP-1 in zebrafish. (a1~a4) imaging of zebrafish incubated in the dark for 1 h in the presence of DCFH-DA and ITBDP-1; (b1~b4) imaging of zebrafish incubated in dark for 1 h in the presence of DCFH-DA and ITBDP-1, and then irradiated (660 nm, 4 mW•cm-2) for 15 min

Figure 11. (a1~a4) AO/EB imaging of A549 cells incubated with ITBDP-1 in dark; (b1~b4) AO/EB imaging of A549 cells incubated with ITBDP-1 under light irradiation for 5 min; (c1~c4) AO/EB imaging of A549 cells incubated with ITBDP-1 under light irradiation for 15 min (excitation wavelength 660 nm, 4 mW•cm-2)

Figure 12. (a1~a4) imaging of A549 cells incubated with ITBDP-2 in dark; (b1~b4) imaging of A549 cells incubated with ITBDP-2 under light irradiation for 15 min; (c1~c4) imaging of A549 cells incubated with ITBDP-2 under light irradiation for 30 min (excitation wavelength 660 nm, 4 mW•cm-2)

Figure 13. (a) Scratch state within 24 h after illumination for 15 min (660 nm, 4 mW•cm-2) in the presence of ITBDP-1 (2 μmol•L-1); (b) scratch state within 24 h in dark in the presence of ITBDP-1 (2 μmol•L-1)

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